Ectopic pigment cells in <i>pde</i> mutants are detectable by 35 hpf, and generated by localised increased proliferation of neural crest-derived cells.

(A-D) Whole mount in situ hybridization of 35 hpf WT (A and C) and pde mutant (B and D) embryos shows ectopic expression in pde mutants (white arrowheads in D) of melanocyte marker dct (A and B) and the iridophore marker ltk (C and D). (E-G) Immunodetection of the proliferation marker phosphohistone 3 (PH3) in neural crest derived cells (labelled with membrane–tethered GFP due to Tg(-4725sox10:cre)ba74; Tg(hsp:loxp-dsRed-loxp-LYN-EGFP)) of 32 hpf WT (E; white arrowhead) and pde mutant (F, white arrowheads) sibling embryos. Quantification of double positive GFP+ PH3+ cells in medial migratory pathway, shows a significant increase in pde mutants compared to WT siblings (Total; WT mean±s.e. = 3.9±0.42, n = 20 and pde = 6.05±0.41, n = 20; p+ PH3 cells in the medial migratory pathway into those dorsal and ventral to the notochord shows that this increase is not detected on the dorsal medial pathway (dorsal double positive cells, WT = 2.35±0.35, n = 20 and pde = 2.85±0.35, n = 20; ppde = 3.2±0.32, n = 20; p<0.0007, two-tailed t-test). Scale bar = 30 μm (A-D) 15 μm (E-F).